Elsevier

Experimental Eye Research

Volume 111, June 2013, Pages 36-49
Experimental Eye Research

Distribution of thrombospondins and their neuronal receptor α2δ1 in the rat retina

https://doi.org/10.1016/j.exer.2013.03.012Get rights and content

Highlights

  • In healthy adult rat retina, TSP1 and TSP2 had an apparent differential distribution.

  • TSP1 was expressed by retinal neurons and TSP2 was expressed by retinal glial cells.

  • In rat retina, both retinal neurons and glial cells expressed α2δ1 receptor.

  • After elevated IOP, TSP2 and α2δ1 were greatly increased with the glial activation.

  • Glia/TSP2-α2δ1 pathway might be involved in synaptic plasticity after retina injury.

Abstract

The role of the extracellular matrix protein thrombospondins (TSPs) in promoting synaptogenesis is gaining more and more attention. The binding of TSP1 and TSP2 to their neuronal receptor α2δ1 stimulates excitatory synaptogenesis in the development and injury of the central nervous system; however, the specific cellular localization and expression of TSP1/2 and α2δ1 in healthy and damaged retinas is unknown. This, to a certain extent, has restricted the progress of research on the molecular mechanisms triggering synaptic plasticity after retinal injury. Here, the cellular localization and expression of TSP1/2 and their receptor α2δ1 was studied in healthy and damaged adult retina induced by elevated intraocular pressure (IOP) using double immunofluorescence labeling and confocal scanning microscopy. We showed the apparent differential distribution of TSP1 and TSP2 in the adult rat retina. TSP1 was confined to the ganglion cell layer and inner nuclear layer, in which it was preferentially expressed by ganglion cells, bipolar cells and horizontal cells but rarely expressed by glial cells. TSP2 staining was diffusely distributed in GFAP- and GS-immunopositive glial cells and processes in the inner retina. In rat retinas, α2δ1 staining was present in ganglion cells, bipolar cells, partial horizontal cells and amacrine cells and the presynaptic terminals. Müller cells and a minority of astrocytes also expressed α2δ1. On the seventh day of elevated IOP, TSP2 immunoreactivity was greatly increased, and immunopositive processes extended throughout the retinal layer and co-localized with GFAP- and GS-positive glial cells. TSP1 distribution in the retina, however, did not change distinctly. α2δ1-immunopositive processes were also increased on the seventh day after elevated IOP. Our study suggested that in the adult rat retina, TSP2, but not TSP1, secreted by glial cells may be involved in the synaptic plastic process after retinal injury through binding to its neuronal receptor α2δ1.

Introduction

The retina is an easily accessible part of the central nervous system (CNS) and often serves as a model system for the study of brain injury and repair. Various injury conditions, including ischemia-reperfusion induced by elevated intraocular pressure (IOP), retinal detachment, and oygen-induced retinopathy (Chen et al., 2008; Sethi et al., 2005; Fu et al., 2011; Dorfman et al., 2011; Dijk et al., 2007), could induce plastic changes of retinal synapses, which may be the direct cause of post-injury visual function impairment. Abnormal synaptic changes may explain why visual function cannot recover as effectively as neuronal survival following many interventions. Therefore, it is necessary to study the protection of synapses and processes that rescue neurons from death following retinal injury and repair; however, the potential molecular mechanisms triggering synaptic plasticity after retinal injury still remain unclear. Glial cells have been shown to participate in the regulation of synaptic transmission and plasticity (Volterra and Meldolesi, 2005; Mauch et al., 2001; Henneberger et al., 2010). Emerging evidences have demonstrated that thrombospondin (TSP) 1 and TSP2 expressed by immature and reactive glial cells were responsible for excitatory CNS synaptogenesis or synapse formation through an interaction with their neuronal receptor, the calcium channel subunit α2δ1 (Christopherson et al., 2005; Eroglu et al., 2009; Procko and Shaham, 2009).

Thrombospondins (TSPs) are large oligomeric, multidomain, extracellular matrix proteins that have been previously shown to play important roles in cell attachment, cell migration, cytoskeletal dynamics and angiogenesis. TSPs mediate these functions via their interactions with various cell surface receptors through specific domains (Tan and Lawler, 2009; Kyriakides and Maclauchlan, 2009). There are five TSPs which are separated into two subfamilies, A and B, according to their overall organization. TSP1 and TSP2 belong to the subgroup A family and are assembled as trimers. They share the same structural and functional domains. It was recently shown that in the developing CNS, TSP1 and TSP2 promoted the formation of new synapses (Christopherson et al., 2005; Crawford et al., 2012). Alpha-2-delta (α2δ) is a membrane-spanning auxiliary protein subunit of voltage-gated calcium channels (VGCCs) that is found in muscle and brain, where it plays an essential role in controlling neurotransmitter release, neuronal excitability and gene expression (Taylor and Garrido, 2008). The α2δ family consists of at least four distinctive genes, α2δ1, α2δ2, α2δ3 and α2δ4. A key feature of α2δ1 is that it is considered to be the high-affinity receptor for two commonly used anti-epileptic drugs, gabapentin and pregabalin. Recently, α2δ1 and α2δ3 have been shown to have novel functions in synaptogenesis independent of their effects on calcium channels (Kurshan et al., 2009; Bauer et al., 2010).

It has also been widely reported that glial cells in the retina were distinctly activated in response to glaucoma, retinal ischemia-reperfusion injury induced by elevated IOP, and photoreceptor degeneration (Middeldorp and Hol, 2011; Lorber et al., 2012; Grosche et al., 1995). It is unknown whether reactive glial cells are responsible for the synaptic plasticity process that is induced by retinal injury through secreting TSPs that interact with α2δ1. We speculated that TSP1/2 is a likely modulator of synaptic plasticity after retinal injury; however, little is known about the specific cellular localization and distribution pattern of TSP1/2 and their receptor α2δ1 in retinal tissue.

In the present study, we identified in detail the immunohistochemical localization of TSP1/2 and their neuronal receptor α2δ1 in adult rat retinas. We also further investigated the changes in TSP1/2 and α2δ1 expression after injury through a retinal ischemia-reperfusion model induced by elevated IOP.

Section snippets

Animals

Fifteen healthy adult Sprague–Dawley rats (no limit of male or female) weighing 200–250 g were acquired in-house from the animal center of Hunan Agricultural University, China (license No. SCXK (Xiang) 2009-0012). The animals were given tap water and food in an environmentally controlled room at a temperature of 25 °C and a relative humidity of 50%–60% with a 12-h:12-h light–dark cycle (light on from 7:00–19:00). The animal protocol was in compliance with the guidelines of the EU Directive

Immunolocalization of TSP1 in the adult rat retina

In the rat retina, TSP1 was greatly distributed in the GCL, where TSP1 immunoreactivity was expressed only in the membrane and cytoplasm of each cell (Fig. 1A). Mild staining was also observed in the cell bodies of the inner nuclear layer (INL). No TSP1 immunoreactivity, however, was visible in the outer nuclear layer (ONL). The pigment epithelial cells exhibited fairly strong dense labeling.

To identify whether TSP1 expression occurred in glial cells in the retina, double immunofluorescence

Discussion

TSP1 and TSP2 have been termed matrix cellular proteins and implicated in a variety of processes, including cell adhesion, cell migration, and developmental and pathological angiogenesis through interacting with several cell surface receptors and other extracellular matrix proteins (Tan and Lawler, 2009). In addition, TSP1 is a major component of platelet a-granules, while TSP2 is typically a product of fibroblasts (Kyriakides and Maclauchlan, 2009). Most notably, evidence has emerged that both

Acknowledgments

This work was sponsored by the National Natural Science Foundation of China, No. 81070729; the Doctoral Foundation of Ministry of Education of China, No. 20100162110067; and the Natural Science Foundation of Hunan Province, No. 10JJ4023 and supported by the Hunan Provincial Innovation Foundation For Postgraduate, No. CX2011B047.

References (43)

  • C.P. Taylor et al.

    Immunostaining of rat brain, spinal cord, sensory neurons and skeletal muscle for calcium channel alpha2-delta (alpha2-delta) type 1 protein

    Neuroscience

    (2008)
  • Y. Wei et al.

    Enhanced protein expressions of sortilin and p75NTR in retina of rat following elevated intraocular pressure-induced retinal ischemia

    Neurosci. Lett.

    (2007)
  • O. Wiser et al.

    The alpha 2/delta subunit of voltage sensitive Ca2+ channels is a single transmembrane extracellular protein which is involved in regulated secretion

    FEBS Lett.

    (1996)
  • M. Adachi et al.

    High intraocular pressure-induced ischemia and reperfusion injury in the optic nerve and retina in rats

    Graefes. Arch. Clin. Exp. Ophthalmol.

    (1996)
  • R.L. Cole et al.

    Differential distribution of voltage-gated calcium channel alpha-2 delta (alpha2delta) subunit mRNA-containing cells in the rat central nervous system and the dorsal root ganglia

    J. Comp. Neurol.

    (2005)
  • D. Chen et al.

    Synaptophysin expression in rat retina following acute high intraocular pressure

    Acta Histochem. Cytochem.

    (2008)
  • D.C. Crawford et al.

    Astrocyte-derived thrombospondins mediate the development of hippocampal presynaptic plasticity in vitro

    J. Neurosci.

    (2012)
  • A. Davies et al.

    The a2d subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational modification essential for function

    Proc. Natl. Acad. Sci. U. S. A.

    (2010)
  • A.L. Dorfman et al.

    Immunohistochemical evidence of synaptic retraction, cytoarchitectural remodeling, and cell death in the inner retina of the rat model of oygen-induced retinopathy (OIR)

    Invest. Ophthalmol. Vis. Sci.

    (2011)
  • Q.L. Fu et al.

    Soluble Nogo-66 receptor prevents synaptic dysfunction and rescues retinal ganglion cell loss in chronic glaucoma

    Invest. Ophthalmol. Vis. Sci.

    (2011)
  • O. Garcia et al.

    A role for thrombospondin-1 deficits in astrocyte-mediated spine and synaptic pathology in Down's syndrome

    PLoS. One

    (2010)
  • Cited by (20)

    • Pregabalin affords retinal neuroprotection in diabetic rats: Suppression of retinal glutamate, microglia cell expression and apoptotic cell death

      2019, Experimental Eye Research
      Citation Excerpt :

      Pregabalin binds to the α2δ-1 and α2δ-2 auxiliary subunits of calcium channels, where α2δ-1 subunit is identified as the high-affinity and the key receptor that mediates the analgesic action of pregabalin (Gee et al., 1996; Patel and Dickenson, 2016). In addition, α2δ-1 is highly expressed by many CNS neurons, including retinal neurons, ganglion cells and glial cells where the receptor expression is greatly increased with the glial activation (Cole et al., 2005; Huang et al., 2013a,b). Pregabalin has a neuroprotective effect against seizures and neuronal excitotoxicity in pilocarpine-induced acute seizures in rats with STZ-induced diabetes and against experimental head trauma in rats (Calikoglu et al., 2015; Huang et al., 2013a,b).

    View all citing articles on Scopus
    View full text